Francesca Reggiani

Complementary Populations of Human Adipose CD34+ Progenitor Cells Promote Growth, Angiogenesis, and Metastasis of Breast Cancer

Obesity is associated with an increased frequency, morbidity, and mortality of several types of neoplastic diseases, including postmenopausal breast cancer. We found that human adipose tissue contains two populations of progenitors with cooperative roles in breast cancer. CD45(-)CD34(+)CD31(+)CD13(-)CCRL2(+) endothelial cells can generate mature endothelial cells and capillaries. Their cancer-promoting effect in the breast was limited in the absence of CD45(-)CD34(+)CD31(-)CD13(+)CD140b(+) mesenchymal progenitors/adipose stromal cells (ASC), which generated pericytes and were more efficient than endothelial cells in promoting local tumor growth. Both endothelial cells and ASCs induced epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells. Endothelial cells (but not ASCs) migrated to lymph nodes and to contralateral nascent breast cancer lesions where they generated new vessels. In vitro and in vivo, endothelial cells were more efficient than ASCs in promoting tumor migration and in inducing metastases. Granulocyte colony-stimulating factor (G-CSF) effectively mobilized endothelial cells (but not ASCs), and the addition of chemotherapy and/or of CXCR4 inhibitors did not increase endothelial cell or ASC blood mobilization. Our findings suggest that adipose tissue progenitor cells cooperate in driving progression and metastatic spread of breast cancer.

The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells

The human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression. The biguanide Metformin (Met), commonly used for Type 2 diabetes, might have activity against BC and was found to inhibit angiogenesis in vivo. We studied Met and another biguanide, phenformin (Phe), in vitro and in vivo in BC models. In vitro, biguanides activated AMPK, inhibited Complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. In coculture, biguanides inhibited the production of several angiogenic proteins. In vivo, biguanides inhibited local and metastatic growth of triple negative and HER2+ BC in immune‐competent and immune‐deficient mice orthotopically injected with BC. Biguanides inhibited local and metastatic BC growth in a genetically engineered murine model model of HER2+ BC. In vivo, biguanides increased pimonidazole binding (but not HIF‐1 expression) of WAT progenitors, reduced tumor microvessel density and altered the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. Phe was significantly more active than Met both in vitro and in vivo. Considering their safety profile, biguanides deserve to be further investigated for BC prevention in high‐risk subjects, in combination with chemo and/or targeted therapy and/or as post‐therapy consolidation or maintenance therapy for the prevention of BC recurrence.

The pan-class i phosphatidyl-inositol-3 kinase inhibitor NVP-BKM120 demonstrates anti-leukemic activity in acute myeloid leukemia

Aberrant activation of the PI3K/Akt/mTOR pathway is a common feature of acute myeloid leukemia (AML) patients contributing to chemoresistance, disease progression and unfavourable outcome. Therefore, inhibition of this pathway may represent a potential therapeutic approach in AML. The aim of this study was to evaluate the pre-clinical activity of NVP-BKM120 (BKM120), a selective pan-class I PI3K inhibitor, on AML cell lines and primary samples. Our results demonstrate that BKM120 abrogates the activity of the PI3K/Akt/mTOR signaling, promoting cell growth arrest and significant apoptosis in a dose- and time-dependent manner in AML cells but not in the normal counterpart. BKM120-induced cytotoxicity is associated with a profound modulation of metabolic behaviour in both cell lines and primary samples. In addition, BKM120 synergizes with the glycolitic inhibitor dichloroacetate enhancing apoptosis induction at lower doses. Finally, in vivo administration of BKM120 to a xenotransplant mouse model of AML significantly inhibited leukemia progression and improved the overall survival of treated mice. Taken together, our findings indicate that BKM120, alone or in combination with other drugs, has a significant anti-leukemic activity supporting its clinical development as a novel therapeutic agent in AML.

Aspirin and atenolol enhance metformin activity against breast cancer by targeting both neoplastic and microenvironment cells

Metformin can induce breast cancer (BC) cell apoptosis and reduce BC local and metastatic growth in preclinical models. Since Metformin is frequently used along with Aspirin or beta-blockers, we investigated the effect of Metformin, Aspirin and the beta-blocker Atenolol in several BC models. In vitro, Aspirin synergized with Metformin in inducing apoptosis of triple negative and endocrine-sensitive BC cells, and in activating AMPK in BC and in white adipose tissue (WAT) progenitors known to cooperate to BC progression. Both Aspirin and Atenolol added to the inhibitory effect of Metformin against complex I of the respiratory chain. In both immune-deficient and immune-competent preclinical models, Atenolol increased Metformin activity against angiogenesis, local and metastatic growth of HER2+ and triple negative BC. Aspirin increased the activity of Metformin only in immune-competent HER2+ BC models. Both Aspirin and Atenolol, when added to Metformin, significantly reduced the endothelial cell component of tumor vessels, whereas pericytes were reduced by the addition of Atenolol but not by the addition of Aspirin. Our data indicate that the addition of Aspirin or of Atenolol to Metformin might be beneficial for BC control, and that this activity is likely due to effects on both BC and microenvironment cells.

Extracellular ATP induces apoptosis through P2X7R activation in acute myeloid leukemia cells but not in normal hematopoietic stem cells

Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2×7R is the most consistently expressed by tumors. P2×7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2×7R. Here, we show that P2×7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2×7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo. Overall, our results demonstrate that P2×7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML.

Adipose progenitor cell secretion of GM-CSF and MMP9 promotes a stromal and immunological microenvironment that supports breast cancer progression

A cell population with progenitor-like phenotype (CD45-CD34+) resident in human white adipose tissue (WAT) is known to promote the progression of local and metastatic breast cancer and angiogenesis. However, the molecular mechanisms of the interaction have not been elucidated. In this study, we identified two proteins that were significantly upregulated in WAT-derived progenitors after coculture with breast cancer: granulocyte macrophage colony-stimulating factor (GM-CSF) and matrix metallopeptidase 9 (MMP9). These proteins were released by WAT progenitors in xenograft and transgenic breast cancer models. GM-CSF was identified as an upstream modulator. Breast cancer-derived GM-CSF induced GM-CSF and MMP9 release from WAT progenitors, and GM-CSF knockdown in breast cancer cells neutralized the protumorigenic activity of WAT progenitors in preclinical models. GM-CSF neutralization in diet-induced obese mice significantly reduced immunosuppression, intratumor vascularization, and local and metastatic breast cancer progression. Similarly, MMP9 inhibition reduced neoplastic angiogenesis and significantly decreased local and metastatic tumor growth. Combined GM-CSF neutralization and MMP9 inhibition synergistically reduced angiogenesis and tumor progression. High-dose metformin inhibited GM-CSF and MMP9 release from WAT progenitors in in vitro and xenograft models. In obese syngeneic mice, metformin treatment mimicked the effects observed with GM-CSF neutralization and MMP9 inhibition, suggesting these proteins as new targets for metformin. These findings support the hypothesis that GM-CSF and MMP9 promote the protumorigenic effect of WAT progenitors on local and metastatic breast cancer. Cancer Res; 77(18); 5169-82. ©2017 AACR.

Abstract 5214: Synergistic activity of aspirin, atenolol and metformin in the inhibition of angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells

We have recently described that the human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression (Martin-Padura et al, 2012; Orecchioni et al, 2013). The biguanide metformin (met), commonly used for type 2 diabetes, might have activity against BC and we found it able to inhibit angiogenesis in vivo (Dallaglio et al, 2014; Orecchioni et al, 2014). We studied met and another biguanide, phenformin (phe), in vitro and in vivo in orthotopic NSG murine models of local and metastatic BC. As met is frequently administered with aspirin or atenolol in diabetic/obese patients, we studied in vitro and vivo their association. In vitro, biguanides activated AMPK, inhibited complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. Aspirin was synergistic with met and phe in inducing apoptosis of estrogen receptor+ BC cells. This synergistic effect was less evident in triple negative BC cells. In co-culture, biguanides significantly inhibited the production of several angiogenic proteins. In vivo, biguanides inhibited local and metastatic growth of triple negative and HER2+ BC in immune-competent and immune-deficient mice orthotopically injected with BC. Biguanides also inhibited local and metastatic BC growth in a genetically engineered model of HER2+ BC. In vivo, biguanides increased pimonidazole binding (but not HIF-1 expression) of WAT progenitors, reduced tumor microvessel density and impaired of the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. This effect was significantly increased by the addition of aspirin or atenolol. This was evident also when AMPK activation was assessed in combination therapy regimens. AMPK phosphorylation was significantly increased in BC cells treated with met+aspirin or phe+atenolol as compared to met or phe alone. In WAT progenitors, AMPK activation was enhanced only under met+aspirin and met+atenolol combinations. In immune-competent mice, met effect in BC models was significantly enhanced by the addition of atenolol or of aspirin. Phe was significantly more active than met both in vitro and in vivo. Considering their safety profile, biguanides (alone or in combination with aspirin or atenolol) deserve to be further investigated for BC prevention in high-risk subjects, in combination with chemo and/or targeted therapy and/or as post-therapy consolidation or maintenance therapy for the prevention of BC recurrence. Citation Format: Giovanna Talarico, Francesca Reggiani, Stefania Orecchioni, Patrizia Mancuso, Angelica Calleri, Giuliana Gregato, Valentina Labanca, Douglas M. Noonan, Katiuscia Dallaglio, Adriana Albini, Francesco Bertolini. Synergistic activity of aspirin, atenolol and metformin in the inhibition of angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5214. doi:10.1158/1538-7445.AM2015-5214

Abstract 5919: Identification of a novel molecular interaction, targeted by Metformin, between breast cancer and white adipose tissue progenitors

The white adipose tissue (WAT) contains a resident population of progenitors with mesenchymal- or endothelial-like phenotype, able to promote breast cancer (BC) progression (Orecchioni et al., 2013). We recently characterized a novel molecular axis underneath the interaction between BC and WAT progenitors, possibly explaining the acquisition of a cancer-associated phenotype by WAT progenitor cells and the establishment of a permissive tumor microenvironment. GM-CSF balance was identified to be the principal regulator of the transcriptional alteration occurring in WAT progenitors exposed to BC: GM-CSF, produced by BC cells, dramatically changed the secretion profile of progenitors, enhancing its own production through a positive regulatory loop and inducing the expression of several factors involved in inflammation (IL-1β), ECM remodeling and angiogenesis (MMP9). IL-1β was strongly down-regulated by GM-CSF neutralization in WAT progenitors in vitro and in preclinical models. Induction of IL-1β by GM-CSF was able, in turn, to up-regulate MMP9 in the same cells. The markedly increased expression of MMP9 was already observed in BC-associated WAT progenitors, being addressed to increased local and metastatic tumor growth in obese mice. GM-CSF production was significalntly enhanced in the presence of concomitant obesity in preclinical models of BC, both in orthotopic models (MMTV-neu/ErbB2; 4T1) and in spontaneous tumor-bearing mice (MMTV-PyMT). GM-CSF neutralization in several obese syngeneic models of BC significantly reduced immunosuppressive cells in tumors and in the surrounding WAT, including T-regs (CD4+CD25brightCD127low), Granulocytic myeloid-derived suppressor cells (G-MDSCs, CD11b+Ly6C-Ly6G+), tumor-associated macrophages (TAMs, F4/80+CD206+MCHIIlow) and tumor infiltrating lymphocytes (TILs, CD8+PD1+). Different BC types produce distinct amounts of GM-CSF: triple negative BC (TNBC) expressed higher levels compared to hormone receptors positive cells, correlating GM-CSF expression to higher tumor grade. In the context of targeting WAT-BC interaction, Metformin was able to impair tumor growth, metastasis and angiogenesis in preclinical models (Orecchioni et al., 2015) and affecting immune cells, including T-regs, TAMs and MDSCs. From our data, GM-CSF was identified to be a new target of Metformin in both BC and WAT progenitors, suggesting a novel inhibitory mechanism mediated by the drug. Metformin displayed the same efficacy in reducing BC growth, metastatic spread and immune system regulation, when compared to GM-CSF specific neutralization in obese syngeneic models. Taken together, these results indicate GM-CSF as a new molecular target of Metformin and support the role of this novel mechanism in BC progression. Ongoing studies also suggest that this pathway might be induced in WAT progenitors by other solid tumors, including non-Hodgkin’s B-lymphoma. Citation Format: Francesca Reggiani, Valentina Labanca, Patrizia Mancuso, Andrea Manconi, Francesco Bertolini. Identification of a novel molecular interaction, targeted by Metformin, between breast cancer and white adipose tissue progenitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5919. doi:10.1158/1538-7445.AM2017-5919

Erratum: The pan-class I phosphatidyl-inositol-3 kinase inhibitor NVP-BKM120 demonstrates anti-leukemic activity in acute myeloid leukemia (Scientific Reports (2015) 5 (18137) DOI: 10.1038/srep18137)

“M.A. designed and performed the research, analyzed data and wrote the manuscript; M.R.R. designed the research, analyzed data and edited the manuscript; S.M. performed metabolic experiments R.L. performed functional assays; S.O., F.R. and G.T. performed in vivo experiments; R.F. provided clinical samples; F.B. designed in vivo research and edited the manuscript; S.A. conceived of the research, provided clinical samples and edited the manuscript; A.T. conceived the research and edited the manuscript. All authors reviewed and approved the manuscript.”

Abstract 3374: GM-CSF and MMP9 are key regulators of the effect of adipose progenitor cells over breast cancer onset and metastatic progression in obesity

We recently described a human cell population with progenitor-like phenotype (CD45-CD34+), resident in the white adipose tissue (WAT) and able to promote local and metastatic breast cancer (BC) progression and angiogenesis (Orecchioni et al., 2013). The molecular mechanism involved in this interaction has been so far elusive. An extensive screening of candidate molecules related to angiogenesis, inflammation, motility and invasiveness revealed that two proteins are significantly up-regulated in WAT-derived progenitors following culture with BC cells: Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Matrix metallopeptidase 9 (MMP9). In vivo, both proteins were overexpressed in orthotopic models of human BC co-injected with human WAT progenitors. The inhibition of GM-CSF by a monoclonal antibody in diet-induced, obese BC mice led to a reduced intratumor vascularization and a strong impairment of WAT immunosuppressive microenvironment, targeting mainly myeloid cells such as macrophages and myeloid derived suppressor cells (MDSCs) in peritumoral WAT. Circulating levels of monocytes and of CD4+CD25brightCD127low/neg T-regulatory cells (T-regs) were also reduced in treated mice. Similarly, soluble immunosuppressive factors, such as IL-10 and IL-5, and CD274 (PD-L1) were reduced in tumors and WAT collected from immune competent mice neutralized for GM-CSF, confirming the crucial role of the factor in promoting tumor immune escape. This resulted in a significantly reduced local BC growth and lower metastatic progression in vivo. All these findings challenge the clinical use of GM-CSF. In the same syngeneic model, MMP9 inhibition reduced neoplastic angiogenesis and significantly decreased local and metastatic tumor growth, without altering immune cells composition in tumor microenvironment. The combined inhibition of GM-CSF and MMP9 was synergic in impairing angiogenesis, local and metastatic BC growth in diet-induced obese orthotopic BC models, indicating a potential complementary role in tumor spread. As we recently reported that Metformin targets both BC cells and the neoplastic WAT environment (Orecchioni et al., 2015), we investigated Metformin effect over GM-CSF and MMP9 expression. Metformin inhibited GM-CSF and MMP9 up-release from WAT progenitors in vitro. Circulating GM-CSF was significantly impaired in BC xenografts administered with Metformin and MMP9 expression was also affected by the treatment. Collectively these results indicate GM-CSF and MMP9 as key candidates involved in the pro-tumorigenic effect of WAT progenitors on BC in a setting of obesity. The comparison between Metformin and GM-CSF/MMP9 specific inhibition is currently under investigation. Citation Format: Francesca Reggiani, Valentina Labanca, Giovanna Talarico, Stefania Orecchioni, Patrizia Mancuso, Francesco Bertolini. GM-CSF and MMP9 are key regulators of the effect of adipose progenitor cells over breast cancer onset and metastatic progression in obesity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3374.